Fibrillar collagens (e.g. collagen type I, II, III, V and XI) consist of a triple helical domain, flanked by telopeptides at both the aminoterminal and carboxyterminal end of the molecule (N-telopeptide and C-telopeptide, respectively). Biosynthesis of collagen is a multistep process, resulting in modifications of both the triple helix and the telopeptides. One of the steps in the biosynthesis of collagen is hydroxylation of certain lysine residues in the triple helix and telopeptides by the enzyme lysyl hydroxylase (EC 1.14.11.4).
Extracellular collagen molecules aggregate spontaneously into microfibrils. Further stabilization of the molecules occurs by means of cross-links. Cross-linking is initiated by conversion of specific lysine (Lys) or hydroxylysine (Hyl) residues of the telopeptides into the aldehydes allysine and hydroxyallysine, respectively, by the enzyme lysyl oxidase (EC 1.4.3.13) [H. M. Kagan, 1994, Path. Res. Pract., 190: 910–919]. The aldehydes subsequently react with Lys or Hyl residues in the triple helix to give characteristic di-functional cross-links. These cross-links eventually mature into tri- or tetra-functional cross-links [D. R. Eyre, 1987, Meth. Enzymol., 144: 115–139; A. J. Bailey et al., 1998, Mech. Ageing Developm, 106: 1–56].
Two related routes for the formation of cross-links have been described, one based on allysine from the telopeptides, the other based on hydroxyallysine from the telopeptides. Each route results in chemically distinct cross-links. Examples of the hydroxyallysine cross-links are hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP); the precursors of these cross-links are di-functional cross-links known in their reduced form as dihydroxylysinonorleucine (DHLNL) and hydroxylysinonorleucine (HLNL), respectively.
It is well known that the stability of collagen molecules in environments containing proteinases depends, amongst others, on the level of cross-linking. The stability of collagen molecules against proteinases can be enhanced by increasing the amount of cross-links. Cross-links can be enzymatically mediated cross-links and non-enzymatically mediated cross-links. The enzymatically mediated cross-links are generated by lysyl oxidase. Introduction of cross-links in a non-enzymatic way can be achieved by treating collagen with a variety of chemicals, such as aldehydes, epoxides, isocyanates, acyl azides, carbodiimides, reducing sugars (the so-called Maillard reaction), or by a variety of physical methods, such as irradition (e.g. short-wave UV irradiation) or dehydrothermal treatments. There is an extensive amount of literature and patents dealing with controlling the biodegradation time of collagen by means of enhancing collagen cross-linking by lysyl oxidase, chemicals or physical methods. Controlling the degradation time of collagenous materials is highly important, especially in the field of drug release and tissue engineering. The starting point to engineer a tissue is the design of a scaffold and a consideration of the kind of cells to be seeded into the scaffold. Scaffolds can also be used in various wound healing situations. Biodegradation of scaffolds is required to prevent longterm physical hindrance of the implant. The rate of degradation is dependent on the application and has to be in concert with tissue formation. Collagen is often used as a basis for the manufacturing of scaffolds. For a number of applications, non-crosslinked collagen cannot be used because of its susceptibility to decomposition by metalloproteinases before it can be remodelled into a resistant replacement. In such cases, a collagen scaffold is needed showing higher resistancies toward proteinases. Therefore, various methods have been developed to control the speed of degradation of collagen, such as the above mentioned chemical and physical methods.
A disadvantage of chemical and physical methods is that the position of the cross-links within the molecule cannot be controlled: cross-links are generated throughout the molecule. In addition, said cross-links can be intramolecular or intermolecular. Another disadvantage is that most chemical and physical methods partially denature the collagen molecules: denatured collagen is highly susceptible to proteolytic degradation. Other disadvantages are that certain cross-links show some toxicity, have immunogenic properties, adversity affect biomechanical properties, adversity affect cell/matrix interactions, or that the treatment enhances unwanted side-effects, such as calcification of the matrix.
These problems can be overcome by using lysyl oxidase: the formed aldehydes react with amino acids located at very specific positions within the triple helix. Furthermore, because the cross-links normally occur in vivo, the cross-links do not show toxicity or immunogenicity, and the treatment with lysyl oxidase does not result in a denaturation of collagen molecules.
In some cases, the durability of collagen molecules cross-linked by means of lysyl oxidase in proteolytic environments is not high enough, resulting in biodegradation times that are too short. In other cases, collagen cross-linked by lysyl oxidase show biodegradation times that are too long. The latter is for example the case in fibrosis. In fibrotic conditions an unwanted accumulation is seen of collagen molecules that is difficult to degrade by proteinases.